Adenylyl cyclases direct the intracellular synthesis of the primary second messenger, cyclic-3′,5′-adenosine monophosphate (cAMP), by converting ATP to cAMP, principally in response to a diverse family of membrane spanning, G-protein coupled receptors, each activated by its own extracellular hormone or protease. Signal transduction for G-protein coupled receptors occurs through a coupled heterotrimeric G protein complex composed of the alpha (Gα), and beta/gamma (Gβγ) subunits. Upon receptor stimulation, Gα exchanges GTP for GDP, dissociates from both Gβγ and the receptor, and proceeds to directly regulate various effectors, including adenylyl cyclase. Multiple families of Gα proteins have been identified, two of which are named for their effects on regulating adenylyl cyclase activity (Gαs family stimulates all adenylyl cyclases, while Gαi family inhibits most but not all of the adenylyl cyclases). Each of these Gα proteins has its own tissue distribution, and subset of coupled receptors, which favors receptor specific regulation of adenylyl cyclase.
Additional studies have suggested other means by which adenylyl cyclase activity may be regulated within tissues. This concept is derived from findings that a number of adenylyl cyclase isoforms exist, each with their own gene locus, distinct set of responses to intracellular signals and unique tissue distribution. To date, nine separate isoforms (Types I-IX) have been characterized, principally from rodents, each with its own regulatory properties and tissue specific distribution.
The structure of adenylyl cyclases has been greatly studied and the putative domains given standard nomenclature. Topographically, the adenylyl cyclase isoforms are similar, having two six-transmembrane spanning regions associated with an intracellular N-terminus, a large cytoplasmic loop (ICD III, more commonly referred to as “C1”) and a large intracellular C-terminus (more commonly referred to as “C2”). The transmembrane region between the N-terminus and the C1 loop is commonly referred to as “M1”. The M1 region has three extracellular domains (ECD I, II and III), two intracellular domains (ICD I and II) and six transmembrane domains (TM I, II, III, V and VI). The region between the C1 loop and the C-terminus is referred to as “M2”. The M2 region has three extracellular domains (ECD IV, V and VI), two intracellular domains (ICD IV and V) and six transmembrane domains (TM VII, VIII, IX, X, XI and XII). The N-terminus is commonly divided into two regions, designated “N1” and “N2”. The large C1 cytoplasmic loop is also divided into two regions, a long “C1a” region and a shorter “C1b” region. Lastly, the C-terminus is divided into a long “C2a” region and a shorter “C2b” region. An extensive discussion of these regions can be found in Broach, et al., WO 95/30012, which is incorporated herein by reference. The amino acid sequence of the C1a and C2a regions are conserved among the different isoforms. On the other hand, the N-terminus, C1b and C2b regions show the most diversity among the various isoforms.
Based on sequence and functional similarities, these isoforms fall into six distinct classes of adenylyl cyclases. Types IV and VI have a wide tissue distribution.
However, Type IX is in a class of its own, being the most divergent of the isoforms and having a ubiquitous tissue distribution.
Diversity in activities, and differences in distribution and prevalence of adenylyl cyclase isoforms, may contribute to tissue specific regulation of cAMP levels. It is expected that by taking advantage of distinct structural and biochemical differences between different adenylyl cyclases, isoform specific or selective modulators can be discovered. This, in conjunction with knowledge of the proportion and distribution of each isoform in select tissues provides a means by which one can develop either tissue specific, or selective pharmacological agents since it is expected that isoform specific modulators would have tissue specificity related to the distribution of that isoform.
Key to the development of selective pharmacological agents is information pertaining to the tissue specific distribution and prevalence of each isoform. To date most of this information is available for isoform mRNA levels in a handful of non-human mammals, although some select mRNA (e.g. Type V) have been measured for many human tissues. Acquiring information on protein isoform distribution in human tissues is considered an important aspect of pharmaceutical research in this area, since this could either strengthen existing target information or point to different isoforms, when compared with mRNA data.
To date, only three full length human adenylyl cyclase isoforms have been cloned: Type II adenylyl cyclase (Stengel, et al., Hum. Genet. 90: 126-130 (1992)), Type VII adenylyl cyclase (Nomura, et al., DNA Research 1:27-35 (1994)) and Type VIII adenylyl cyclase (Defer, et al., FEBS Letters 351:109-113 (1994)).
Type IX, first cloned from mouse brain, is in a unique isoform class, being the most divergent of the isoforms and having a wide tissue distribution. Premont, et al. Jour. Biol. Chem. 271(23):13900-13907 (1996). The human isoform has not been cloned until now.